Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.
In: Nucleic acids research, Jg. 46 (2018-05-04), Heft 8, S. 4164-4175
academicJournal
Zugriff:
Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).
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Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.
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Autor/in / Beteiligte Person: | Uson, ML ; Carl, A ; Goldgur, Y ; Shuman, S |
Zeitschrift: | Nucleic acids research, Jg. 46 (2018-05-04), Heft 8, S. 4164-4175 |
Veröffentlichung: | 1992- : Oxford : Oxford University Press ; <i>Original Publication</i>: London, Information Retrieval ltd., 2018 |
Medientyp: | academicJournal |
ISSN: | 1362-4962 (electronic) |
DOI: | 10.1093/nar/gky238 |
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